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2 protocols using anti cd28 fitc

1

Foxp3+ Regulatory T Cell Analysis

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For analysis of sorted Foxp3+ Tregs, cells were
stained with anti-CD28-FITC, anti-CD4-PerCp Cy5.5, and
anti-Foxp3-allophycocyanin (all from eBioscience). Intracellular staining was
done after surface staining using a Foxp3 intracellular staining kit
(eBioscience). To determine total numbers of Foxp3+ Tregs,
the number of spleen cells was multiplied by the total percentage of
CD4+Foxp3+ cells detected by flow cytometry. Flow
cytometry was done using a Cyan ADP flow cytometer (Beckman Coulter) and data
analyzed using FlowJo (TreeStar Inc). For assessment of CD4+and CD8+ T cells in spleens of antibody treated mice or
separated T cell subsets, cells were stained with PE-anti-CD4 (RMA-4;
eBioscience or Biolegend) or PE anti-CD8 (53.6; eBioscience or Biolegend).
Samples were analyzed on a FACSCalibur (BD Bioscience) and data analyzed using
FlowJo.
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2

Phenotyping of Immune Cell Subsets

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After thawing PBMCs in PBS containing the endonuclease benzonase (Novagen/Merck Milipore Schwalbach, Germany), cells were directly stained in PBEA buffer (PBS, 0.5% BSA, 2 mM EDTA and 0.01% NaN3 ) for 30 min on 4°C. An overview of the B cell-, T cell- and NK cell/regulatory T cell (Treg)-phenotyping panels including fluorochromes is provided in Table S2. Cells were washed with PBEA buffer and fixed in PBS containing 1% formaldehyde. The following monoclonal antibodies were used after initial testing and titration: anti-CD3 Alexa-eFluor 780, anti-CD4 PE-Cy7, anti-CD19 Pe-Cy7, anti-CD27 APC, anti-CD28 FITC, and anti-CCR7 PE (eBioscience, SanDiego, USA), anti-CD4 PerCP-Cy5.5, anti-CD8+ PerCP-Cy5.5, anti-CD16 PerCP-Cy5.5, anti-CD20 FITC, anti-CD25 V450, anti-CD38 PerCP-Cy5.5, anti-CD45RA HV450, anti-CD69 FITC, anti-CD86 V450, anti-IFN-g APC, anti-IgD PE, anti-TNF-a FITC (Becton Dickenson GmbH, Heidelberg, Germany), anti-IL-2 PacificBlue (BioLegend,); anti-CD56 PE (Miltenyi, Bergisch Gladbach, Germany).
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